Specific fluorometric detection method for cyanide



United States Patent 3,432,269 SPECIFIC FLUOROMETRIC DETECTION METHODFOR CYANIDE George G. Guilbault, Harford County, and David N. Kramer,Baltimore County, Md., assignors to the United States of America asrepresented by the Secretary of the Army No Drawing. Filed Apr. 7, 1966,Ser. No. 541,022 US. Cl. 23-230 9 Claims Int. Cl. G01n 31/22 Thisinvention relates to an improved detection process and more particularlyto a direct process for the specific detection of small quantities ofcyanide.

It is well known in the art that numerous procedures are available forthe estimation of cyanide. These methods may be classified as follows.

(1) N on-Colorimetric Methods:

(a) Titrimetric methods involving visual end point detection (b)Titrimetric methods involving instrumental end point determination (c)Polarographic methods (d) Gas Chromatography (2) Colorimetric Methods:

(a) Methods involving formation of a metal complex (b) Colorimetricmethods based on the Konig reaction.

The above cited methods are not truly specific for the cyanide ion. TheKonig synthesis (the reaction of cyanogen bromide or chloride withpyridine and an aromatic amine to form a dye) permit the determinationof cyanide directly on the original simple and are generally consideredthe best for small amounts of cyanide. 'I he methods involve the use ofcomplicated procedures of more than three process steps and/or involvethe use of carcinogens.

An object of this invention is to provide a simple, direct method forthe detection of a small quantity of cyanide.

Another object of this invention is to determine the quantity of cyanidepresent in the solution after its detection.

Another object of our invention is to develop a onestep method forspecifically detecting sub-microgram quantities of cyanide.

Another object of this invention is to add as little as possible, suchas 0.5 mg. of the unknown solution to a reagent and solvent wherein ahighly green fluorescent product is produced.

The foregoing objects and others are accomplished in accordance withthis invention, generally speaking, by applying the method and employingreagent grade chemicals, pure solvents and triply distilled water. Allanions tested are added in the form of the chemical pure sodium orpotassium salt. A number of quinones and quinone derivatives wereinvestigated to improve the sensitivity namely p-benzoquinone;N-chloro-p-benzoquinoneimine and substituted quinone monoxime benzenesulfonate esters. The order of the rate of reaction and sensitivityincreases in the general order: O-(p-nitro benzene sulfonyl) quinonemonoxime Nchloro-p-benzoquinoneimine p-benzoquinone.

A number of related sulfonate esters were prepared in an attempt toimprove the sensitivity of detection and the results obtained areindicated in Table I. In this table, some of the properties of the finalgreen fluorescent product (wavelength of excitation and emission andfluorescence maximum) as well as the rate of reaction as indicated bythe rate of production of fluorescence with time, AF/At, and the lowestdetectable limit of cyanide (in ,ug. per ml. of total solution used) aregiven.

All fluorescent measurements are made with an Aminco- BowmanSpectrophotofiuorometer, (SPF) equipped with a thermoelectric cooler tomaintain a constant temperature of 25 C.

The various substituted benzene sulfonate esters of quinone monoxime areprepared by reacting equal molar quantities of the sodium salts ofp-nitrosophenol or omethyl-p-nitrosophenol and p-bromo, p-nitro,p-methoxy 0r p-methyl benzene sulfonyl chloride in tetrahydrofuran for15 minutes and adding pyridine with continuous stirring for one hour.The product is then isolated by pouring the reaction mixture into icewater. Filter the precipitate, dry and recrystallize from carbontetrachloride. Analysis and properties of these compounds may be foundin Tables I and 11. These substituted benzene sulfonate esters ofquinone monoxime can be illustrated by the following formula:

Where R is H, or methyl and R is a group such as methyl, nitro,rnethoxy, and bromine, this process is substantially that of Bur-mistrovet al.

Ukain. Khim. Zhur. 22, 2238 (1956). See Chemical Abstracts 51: col.272i.

A series of novel derivatives of the quinone monoxime benzene sulfonateesters are prepared (Tables I and II), in which various substituents areplaced on the quinone and aryl sulfonate rings. In general, all theproducts have approximately the same excitation and emissionwavelengths, 400 to 500 mu, respectively. One might also expect, thesame total fluorescence, since the same product I is believed produced.Evidently, when an electron Withdrawing group is present on thetosylate, a side reaction occurs, namely the alkaline hydrolysis of thetosylate to the quinone monoxime. This compound has been experimentallyshown not to react with cyanide to yield the fluorescent product I.Hence, the total fluorescence observed is less when R is NO or Br (TableI) Substitution on the quinone ring decreased the fluorescence of theproduct, as well as the rate of reaction. Also, the excitation andemission wavelengths were shifted to shorter wavelengths when R groupsin which R is H or lower alkyl are placed on the ring. This ispresumedly due to the inhibition of the 1,4 addition of the secondcyanide to Compound III in forming Compound IV due to the presence ofthe R group as disclosed in Ex. 1.

TABLE I.REACTION OF VARIOUS COMPOUNDS WITH CYANIDE ION [Substrate=3.4 Min DMSO; CN-=3X10- M] Properties of the Lowest Product DetectabloCompound x ex.: m A em.: m Limit of Fm. AF/min., ON, microunitsF.U./min. grains/ml.

r o =C 0 400-420 480490 90 as o. 2

l 11 0 =C 0 41mm 410 8.0 o. 2

m O= =NCl 440 500 75 60 1.0

1v o= =Noso.-@ 440 500 60 33.6 1.0

v 0 =N OSOz@Br 430440 500 as. a 1. 0

vI o= =NOSOr-@NO2 450 500 30 37.8 1.0

v11 O=C =NOSOZ@OCH3 440 500 so 20 1.0

VIII 0 =C =NOSOz-@CH; 430 140 500 so 22. 2 1.0

IX 0 =C =NOSO2@CH3 420 490 23 3. 0 2.0

x o ==Nosoz@-o on, 410420 490 so 10 1.0

TABLE II.-ANALYTICAL DATA ON VARIOUS COMPOUNDS PREPARED AS SUBSTRATESFOR ON- Melting Carbon Hydrogen Nitrogen point, Cale. Other Found C.Cale. Found Cale. Found Cale. Found Compound:

42. l 42. 4 2. 34 2. 3 23. 4 Bromine. 23.6 46. 7 46. 4 2. 6 2. 5 9. 099. 0

A number of different solvents were tested for use in this reaction.However, only two solvents produced the desired results with the orderof fluorescence intensity and rate of reaction increased in the orderdimethylformamide (DMF) dimethylsulfoxide (DMSO).

The effect of solvent on the fluorescence is observed, the totalfluorescence increasing with increasing dielectric constant. This effectshould be observed, since the fluorescent species is an ionic moleculeand solvents favoring the ionic form should be best.

Since the rate and maximum fiourescence is obtained in dimethylsulfoxidethis solvent is used in all determinations with p-benzoquinone.

Over 30 anions were tested for possible reaction with p-benzoquinone,quinone monoxime benzene sulfonate ester, and N-chloro-p-quinoneimine,to give a fluorescent product. The ions tested were iodide, chloride,bromide, hypochlorite, chlorate, perchlorate, iodate, phthalate,silicate, sulfate, bisulfate, thiocyanate, tartrate, nitrate, fiuor ide,phosphate, thiosulfate, citrate, bromate, sulfide, periodate, arsenite,sulfite, ferricyanide, cyanate, carbon ate, bicarbonate, borate,perborate, bismuthate, tellurite, dichromate, ferrocyanide, sulfide,tungstate, arsenate, molybdate, various nitriles and isocyanates. Onlycyanide was found to react with the compounds producing a greenfluorescence. Hence, the method is highly specific for cyanide.

The effect of these various anions is tested on the quantitativedetermination of cyanide. The cyanide used is at a concentration of3X10* M, the substrates p-benzoquinone and quinone monoxime benzenesulfonate ester have a concentration of 3.4 10 M in dimethysulfoxide.All anions are added at a concentration of 0.1 M, their overallconcentration being 3 l0- M in aqueous solution. The following ions hadno effect at the above added concentration: chlorate, perchlorate,nitrate, iodide, iodate, bromide, bismuthate, sulfate, tartrate,fluoride, chloride, phosphite, citrate, phthalate and thiocyanate. Theions phosphate, bicarbonate, molybdate, cyanate, carbonate, borate,perborate, thiosulfate, bismuthate, tungstate, arsenate, sulfite,silicate, bromate, ferricyanide, dichromate, and sulfide interfer in thedetermination due to a pH effect. When these compounds are added in pH7.5 phosphate butler, no interference is observed in the determination.

The eflfect of pH of the solution added is given in Table III. If the pHof the added solution (0.1 ml.) is held at 6.5 to 7.5, the reaction willproceed smoothly.

More acidic or basic conditions will tend to slow the reaction. At pHsof 8-10, the solution will turn yellow due to the occurrence of sidereactions and no fluorescence is observed.

The cyanide may be added in a water solution (Table III) provided basicinterferences are known to be absent (silicate, phophate, etc.). Bestresults are obtained at pH 7.5 using phosphate bulfer and it isrecommended that the sample be prepared in this medium if possible.Water has a deleterious effect on the rate of reaction at high amounts(0.3 ml.), the rate decreasing with increasing quantities of thissolvent (Table III). For this reason, the sample is added in 0.1 ml. ofsolution.

The process of our invention provides for a specific fluorometric methodfor the detection of cyanide. In particular, the technique employedencompasses the addition of the unknown solution to a substrate orreagent such as a quinone monoxime benzene sulfonate ester,N-chlorop-benzoquinoneimine, or p-benzoquinone with a solvent such asdimethylformamide and dimethylsulfoxide.

The fluorescence produced in the reaction of one of the reagents withcyanide, as Well as the initial rate, AF/At, is proportional to thecyanide concentration of the range of concentrations of 0.2 to 50 ng.per ml. of solution. From calibration plots of AF/ At or fluorescence(maximum produced in 3 minutes) vs. cyanide concentration, the amount ofcyanide present may be determined with a deviation of about 5%. However,the reagent p-benzoquinone appears to be the most satisfactory substratefor the detection and determination of cyanide, based on sensitivity(Table I), reproducibilty and accessibility.

The invention is further illustrated by the following examples:

Example 1 To 3 ml. of a 3.4 10 M solution of quinone monoxime benzenesulfonate ester in dimethyl sulfoxide is added 0.1 m1. of the solutionto be analyzed, contained in phosphate buffer, pH 6.5-7.5 and containing0.6 to 150 ,ug. of cyanide. When a green fluorescence develops, this isan indication that cynide is present. This green fluorescence is read inan Aminco-Bowman Spectrophotofluorometer at excitation and emissionwavelengths of 400 to 500 mu, respectively. The amount of cyanidepresent in the solution may be calculated from calibration plots offluorescence readings vs. cyanide concentration.

The reaction of Example 1 is shown as follows:

R R l H CH CN NHOSOt bR' NHOSOz bR' 1v IVA ON ON v VA R is H and loweralkl. R is CH H, 0CH and halogen.

TABLE LIL-EFFECT OF DH AND WATER ON THE REACTION OF p- BENZOQUINONE WITHOYANIDE 1 Total water=0.1 ml. 2 Total water=0.2 ml. 3 Total water=0.3ml. l Total water=0.5 m1.

Example 2 To 3 ml. of 3.4x l0 M solution of p-benzoquinone in dimethylsulfoxide is added 0.1 ml. of the solution to be analyzed, contained inphosphate bufler, pH 6.5-7.5 and containing 0.6 to g. of cyanide. When agreen fluorescence develops, this is an indication that cyanide ispresent. This green fluorescence is read in an Aminco- BowmanSpectrophotofluorometer at excitation and emission wavelengths of 400 to500 mu, respectively. The amount of cyanide present in the solution maybe calculated from calibration plots of fluorescence readings s. cyanideconcentration.

The reaction of Example 2 is shown as follows:

ON CN VI VII The process of this invention may be employed in thespecific detection of cyanide employing various quinone derivatives inconjunction with various solvents. This method is specific for cyanides.It is sensitiveone part per 200 billon of cyanide is detectable. It isfast and an analysis can be performed in two minutes. All the compoundsdisclosed and employed in this process produce a green fluorescence.

While We have illustrated and described the preferred embodiments of ourinvention, it is to be understood that we do not limit ourself to theprecise limitations herein disclosed and that various changes andmodifications may be made within the scope of the invention as definedin the appended claims.

We claim:

'1. A specific fluorometric method for the detection of sub-microgramquantities of cyanide which comprises:

#(a) adding an unknown solution contained in a phosphate buffer andhaving a pH of 6.5 to 7.5 and a concentration up to 0. 1 M to a solutionof a reagent selected from the group consisting of p-benzoquinone,N-chloro-p-benzoquinoneimine, 2,5-dich1oro-4-benzoquinone and asubstituted quinone monoxime represented by the formula:

R is selected from the group consisting of H or CH and R' is selectedfrom the group consisting of CH H, N0 OCH and bromine and a solventtaken from the group consisting of dimethylformamide anddimethylsulfoxide.

(b) registering the developed green fluorescent solution at excitationand emission wavelengths of 400 to 500 mu, respectively,

(0) recording the calibration plots of fluorescence readings versuscyanide concentration whereby the unknown concentration of cyanide maybe determined.

2. A specific fiuorometric method for the detection of cyanide asdefined in claim 1 employing the solvent dimethylsulfoxide and thereagent O-(p-nitro benzene sulfonyl) quinone monoxime.

3. A method as defined in claim 2 employing the solventdimethylformamide.

4. A method as defined in claim .1 employing the solventdimethylsulfoxide and the reagent N-chloro-p-benzoquinoneimine.

5. A method as defined in claim 4 employing the solventdimethylformamide.

6. A method as defined in claim 1 employing the solventdimethylsulfoxide and the reagent p-benzoquinone.

7. A method as defined in claim 6 employing the solventdimethylformamide.

8. A method as defined in claim 1 wherein a concentration of 3X10 M ofthe unknown solution is added to a concentration of a 3.4x1O M solutionof p-benzoquinone in dimethylsulfoxide.

9. A method as defined in claim 8 employing the solventdimethylformamide.

References Cited MORRIS O. WOLK, Primary Examiner.

E. A. KATZ, Assistant Examiner.

'U.S. 'Cl. X.R. 252-408

1. A SPECIFIC FLUOROMETRIC METHOD FOR THE DETECTION OF SUB-MICROGRAMQUANTITIES OF CYANIDE WHICH COMPRISES: (A) ADDING AN UNKNOWN SOLUTIONCONTAINED IN A PHOSPHATE BUFFER AND HAVING A PH OF 6.5 TO 7.5 AND ACONCENTRATION UP TO 0.1 M TO A SOLUTION OF A REAGENT SELECTED FROM THEGROUP CONSISTING OF P-BENZOQUINONE, N-CHLORO-P-BENZOQUINONEIMINE,2,5-DICHLORO-4-BENZOQUINONE AND A SUBSTITUTED QUINONE MONOZIMEREPRESENTED BY THE FORMULA: